Method of assaying type III collagen degradation

ABSTRACT

A method of assaying collagen fragments in body fluids, including bringing a sample of body fluid in contact with at least one immunological binding partner for the collagen fragments, said binding partner being immunoreactive with synthetic peptides, the sequences of which are essentially derived from collagen and containing potential sites for cross-linking. The immunological binding partners are incorporated, either as whole antibodies or as immunologically active fragments thereof, in an assay for quantitative determination of collagen fragments in the sample. In addition to being contacted with the immunological binding partner(s), the sample may be brought into direct contact with the corresponding synthetic peptide. The invention further comprises a test kit and specific means for carrying out the method. The structure of specific peptides is also described.

[0001] The present invention relates to a method of determining collagenfragments in body fluids. The invention further relates to means,including synthetic peptides, monoclonal and polyclonal antibodies andcell lines, for use in carrying out the method of the invention. Stillfurther, the invention relates to the use of the above method todiagnose the presence of disorders associated with the metabolism ofcollagen, especially osteoporosis.

BACKGROUND OF THE INVENTION

[0002] Collagens and Disorders of Collagen Metabolism

[0003] Osteoporosis is the most common bone disease in humans. Primaryosteoporosis, accompanied by increased susceptibility to fractures,results from a progressive reduction in skeletal bone mass. It isestimated to affect 15-20 million individuals in the USA alone. Itsbasis is an age-dependent imbalance in bone remodeling, i.e., in therates of formation and resorption of bone tissue.

[0004] In the USA about 1.2 million osteoporosis-related fractures occurin the elderly each year including about 538,000 compression fracturesof the spine, about 227,000 hip fractures and a substantial number ofearly fractured peripheral bones. Between 12 and 20% of the hipfractures are fatal because they cause severe trauma and bleeding, andhalf of the surviving patients require nursing home care. Total costsfrom osteoporosis-related injuries now amount to at least $10 billionannually in the USA (Riggs, New Enqland Journal of Medicine 327:620-627(1992)).

[0005] Osteoporosis is most common in postmenopausal women who, onaverage, lose 15% of their bone mass in the 10 years after menopause.This disease also occurs in men as they get older and in youngamenorrheic women athletes. Despite the major, and growing, social andeconomic consequences of osteoporosis, the availability of reliableassays for measuring bone resorption rates in patients or in healthysubjects is very limited. Other disorders entailing (and correlatedwith) abnormalities in collagen metabolism include Paget's disease,Marfan's syndrome, osteogenesis imperfecta neoplastic growth incollagenous tissue, dwarfism, rheumatoid arthritis, osteoarthritis andvasculitis syndrome.

[0006] Three known classes of human collagen have been described todate. The Class I collagens, subdivided into types I, II, III, V, andXI, are known to form fibrils. Their full amino-acid sequence (to theextent they have been elucidated) are attached in Appendix A.

[0007] Collagen type I accounts for more than 90% of the organic matrixof bone. Therefore, in principle, it is possible to estimate the rate ofbone resorption by monitoring the degradation of collagen type I.Likewise, a number of other disease states involving connective tissuecan be monitored by determining the degradation of collagen. Examplesare collagen type II degradation associated with rheumatoid arthritisand osteoarthritis and collagen type III degradation in vasculitissyndrome.

[0008] Amino acid sequences of human type III collagen, human pro 1(II)collagen, and the entire prepro 1(III) chain of human type III collagenand corresponding cDNA clones have been investigated and determined byseveral groups of researchers; see Loil et al., Nucleic Acids Research12:9383-9394 (1984); Sangiorgi et al., Nucleic Acids Research13:2207-2225 (1985); Baldwin et al., Biochem J. 262:521-528 (1989); andAla-Kokko et al., Biochem. J. 260:509-516 (1989).

[0009] Type I, II, and III collagens are all formed in the organism asprocollagen molecules, comprising N-terminal and C-terminal propeptidesequences, which are attached to the core collagen molecules. Afterremoval of the propeptides, which occur naturally in vivo duringcollagen synthesis, the remaining core of the collagen moleculesconsists largely of a triple-helical domain having terminal telopeptidesequences which are non-triple-helical. These telopeptide sequences havean important function as sites of intermolecular cross-linking ofcollagen fibrils extracellularly. The alpha-helical region also includescrosslinkable sites. Peptides from this region are part of the presentinvention.

[0010] Intermolecular cross-links provide collagen fibrils withbiomechanical stability. The formation of these cross-links is initiatedby modification of lysine and hydroxylysine residues to thecorresponding aldehydes. Several of these residues located on adjacentchains of collagen will spontaneously form different intermolecularcross-links. The exact position of the sites for cross-linking oncollagen telopeptides and from the helical region has been previouslydescribed. See, for example, Kuhn, K., in Immunochemistry of theExtracellular Matrix 1:1-29, CRC Press, Inc., Boca Raton, Fla. (1982),Eyre, D. R., Ann. Rev. Biochem. 53:717-48 (1984) or U.S. Pat. No.5,140,103). Furthermore, the amino acid sequences of some potentialsites for cross-linking in type I, II, and III collagen are given inTable 1 below.

[0011] The fibrous proteins, collagen and elastin, are cross-linked by aunique mechanism based on aldehyde formation from lysine orhydroxylysine side chains. Four homologous loci of cross-linking areevident in molecules of type I, II and III collagens (for review seeKuhn, K., in Immunochemistry of the Extracellular Matrix 1:1-29 (1982)).Two are aldehyde sites, one in each telopeptide region. The other twosites are hydroxylysine symmetrically placed at about 90 residues fromeach end of the molecule. When collagen molecules pack into fibrils,these latter sites in the helical region align and react withtelopeptide aldehydes in adjacent molecules. There is now strongevidence that 3-hydroxypyridinium residues are the mature cross-linkcoming from hydroxylysine-derived aldehydes. The mature cross-linkingresidues of the other pathway, i.e. from aldehyde formation of lysineresidues, is, however, still unknown.

[0012] Prior Art Assays for Collagen Degradation

[0013] In the past, assays have been developed for monitoringdegradation of collagen in vivo by measuring various biochemicalmarkers, some of which have been degradation products of collagen.However, none of these methods are based upon the use of immunologicalbinding partners in the form of antibodies which are immunoreactive withsynthetic peptides having a sequence essentially derived from collagenfragments having crosslinkable sites.

[0014] For example, hydroxyproline, an amino acid largely restricted tocollagen, and the principal structural protein in bone and all otherconnective tissues, is excreted in urine. Its excretion rate is known tobe increased in certain conditions, notably Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased, as discussedfurther below.

[0015] For this reason, urinary hydroxyproline has been used extensivelyas an amino acid marker for collagen degradation; Singer, F. R. et al.,Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.),489-575 (1978), Academic Press, New York.

[0016] U.S. Pat. No. 3,600,132 discloses a process for the determinationof hydroxyproline in body fluids such as serum, urine, lumbar fluid andother intercellular fluids in order to monitor deviations in collagenmetabolism. The patent states that hydroxyproline correlates withincreased collagen anabolism or catabolism associated with pathologicalconditions such as Paget's disease, Marfan's syndrome, osteogenesisimperfecta, neoplastic growth in collagen tissues and in various formsof dwarfism.

[0017] Bone resorption associated with Paget's disease has also beenmonitored by measuring small peptides containing hydroxyproline, whichare excreted in the urine following degradation of bone collagen;Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5:255-262 (1981),and Singer, F. R., et al., supra.

[0018] In the case of Paget's disease, the increased urinaryhydroxyproline probably comes largely from bone degradation;hydroxyproline, however, generally cannot be used as a specific indexfor bone degradation. Much of the hydroxyproline in urine may come fromnew collagen synthesis (considerable amounts of the newly made proteinare degraded and excreted without ever becoming incorporated into tissuefabric), and from turnover of certain blood proteins as well as otherproteins that contain hydroxyproline.

[0019] Furthermore, about 80% of the free hydroxyproline derived fromprotein degradation is metabolized in the liver and never appears in theurine. Kiviriko, K. I. , Int. Rev. Connect. Tissue Res. 5:93 (1970), andWeiss, P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyprolineis a good marker for osteoporosis, but it is troublesome to handle. Itis specific for collagen in bones.

[0020] Hydroxylysine and its glycoside derivatives, both peculiar tocollagenous proteins, have been considered to be more accurate thanhydroxyproline as markers of collagen degradation. However, for the samereasons described above for hydroxyproline, hydroxylysine and itsglycosides are probably equally non-specific markers of bone resorption;Krane, S. M. and Simon, L. S. Develop. Biochem. 22:185 (1981).

[0021] Other researchers have measured the cross-linking compound3-hydroxypyridinium in urine as an index of collagen degradation injoint diseases. See, for background and as examples, Wu and Eyre,Biochemistry 23:1850 (1984); Black et al., Annals of the RheumaticDiseases 48:641-644 (1989); Robins et al.; Annals of the RheumaticDiseases 45:969-973 (1986); and Seibel et al., The Journal ofDermatology 16:964 (1989). In contrast to the present invention, theseprior researchers have hydrolyzed peptides from body fluids and thenlooked for the presence of individual 3-hydroxypyridinium residues.

[0022] Assays for determination of the degradation of type I, II, andIII collagen are disclosed in U.S. Pat. Nos. 4,973,666 and 5,140,103.However, both these patents are restricted to collagen fragmentscontaining the cross-linker 3-hydroxypyridinium, whereas the presentinvention does not rely on the presence or absence of this particularcross-linking structure. Furthermore, the above-mentioned assays requiretedious and complicated purifications from urine of collagen fragmentscontaining 3-hydroxypyridinium to be used for the production ofantibodies and for antigens in the assays.

[0023] At present very few clinical data using the approach described inU.S. Pat. Nos. 4,973,666 and 5,140,103 are available. Particularly, nodata concerning the correlation between the urinary concentration (asdetermined by methods described in the above-mentioned patents) of3-hydroxypyridinium containing telopeptides of type I collagen and theactual bone loss (as determined by repeated measurements by bonedensitometry) are published. The presence of 3-hydroxypyridiniumcontaining telopeptides in urine requires the proper formation in bonetissue of this specific cross-linking structure at various times beforethe bone resorbing process. Very little information on these processesis available and the present invention seeks to circumvent thisdependance of the correct formation of the cross-linking structure.Furthermore, preliminary data indicate that in one embodiment of thepresent invention a major fraction of the molecules reactive in theassay has a molecular weight of more than 4,000 daltons. On thecontrary, only molecules with a molecular weight below 2,000 daltons areidentified in urine with the monoclonal antibody used in the assay;Hanson et al., Journal of Bone and Mineral Research 7:1251-1258 (1992).This demonstrates that the method according to the present invention hasa very different profile of reactivities, i.e. it detects very differentmolecules, compared to methods described in the two above-mentioned U.S.patents.

[0024] None of the above researchers have reported specifically assayinga crosslinkable collagen fragment that is naturally produced in vivoupon collagen degradation, as in the present invention.

[0025] GB patent application No. 2,205,643 reports that the degradationof type III collagen in the body can be quantitatively determined bymeasuring the concentration of an N-terminal telopeptide from type IIIcollagen in a body fluid. This method does not relate to methodsemploying antibodies reactive with specific, low molecular weightsequences around crosslinkable structures. Instead, the method usesantibodies generated to N-terminal telopeptides released by bacterialcollagenase degradation of type III collagen, said telopeptides beinglabelled and used in the assay.

[0026] There are a number of reports indicating that collagendegradation can be measured by quantitating certain procollagenpeptides. Propeptides are distinguished from telopeptides andalpha-helical region of the collagen core by their location in theprocollagen molecule and the timing of their cleavage in vivo; see U.S.Pat. Nos. 4,504,587; 4,312,853; Pierard et al., Analytical Biochemistry141:127-136 (1984); Niemela, Clin. Chem. 31/8:1301-1304 (1985); andRohde et al., European Journal of Clinical Investigation 9:451-459(1979).

[0027] U.S. Pat. No. 4,778,768 relates to a method of determiningchanges occurring in articular cartilage involving quantifyingproteoglycan monomers or antigenic fragments thereof in a synovial fluidsample. This US patent does not relate to detecting collagen fragmentsderived from degraded collagen.

[0028] Dodge, J. Clin, Invest. 83:647-661 (1981) discloses methods foranalyzing type II collagen degradation utilizing a polyclonal antiserumthat specifically reacts with unwound alpha-chains and cyanogenbromide-derived peptides of human and bovine type II collagens. Contraryto the present invention the degradation products of collagen were notdetected in a body fluid, but histochemically by staining of cellcultures, i.e. by “in situ” detection. The main difference between Dodgeand the present invention is that Dodge measures type II collagendegradation in situ. By “in situ” is meant a determination carried outin the cells or tissue in which the degradation takes place. There isquite a fundamental difference between this determination and a methodbased upon tracing a marker in vitro, e.g. in the urine.

[0029] None of these references specify the structures of particulartelopeptide or alpha-helical degradation products that could be measuredto determine the amount of degraded fibrillar collagen in vivo.

SUMMARY OF THE INVENTION

[0030] The present invention is based on the discovery of the presenceof particular collagen fragments in body fluids of patients and normalhuman subjects. The collagen fragments are generated upon collagendegradation and are partly characterized by the presence of potentialsites for cross-linking, e.g. by the presence of lysine or hydroxylysine(Kühn, K., Immunochemistry of the Extracellular Matrix 1:1-29 (1982)).It is believed that a fraction of the collagen fragments measured by theassays embodied in the present invention are normally covalently linkedin vivo to other peptide fragments through different, unidentified oralready identified cross-links.

[0031] The method of the present invention may be used for determinationof the degradation of human collagen of type I, II, and III.

[0032] The present invention provides a method of assessing thedegradation of collagen based on a determination of the presence andquantity of a particular class of collagen fragments produced in vivoupon collagen degradation; and a comparison of the detected collagenfragments to those of a predetermined standard developed by measuringthe same class of collagen fragments in normal individuals, i.e.individuals not afflicted with a disorder affecting collagen metabolism,said individuals being sex- and age-matched with the subjects beingtested.

[0033] The present invention uses antibodies immunoreactive withsynthetic peptides without these cross-linking structures. Accordingly,it is believed that collagen fragments (corresponding to the syntheticpeptides) with or without actual cross-links, but with crosslinkablesites, are measured in the assays embodied in the present invention.

[0034] In a preferred embodiment, the method is based on the competitivebinding of collagen fragments in body fluids and of synthetic peptidesessentially derived from collagen to immunological binding partners.

[0035] The present invention provides new and very simple procedures forthe detection (qualitative and quantitative) of collagen fragmentsgenerated upon collagen degradation.

[0036] For purposes of the present invention, as disclosed and claimedherein, the following terms are as defined below:

[0037] “Antibody”: A monoclonal or polyclonal antibody or immunoreactivefragment thereof (i.e. capable of binding the same antigenicdeterminant), including—but not limited to—Fab, Fab′, and F(ab′)2fragments.

[0038] “Crosslinkable sites”: loci in collagen telopeptide or helixamino acid sequences containing lysine or hydroxylysine residues whichcan form cross-links with telopeptides or helical amino acid sequencesof other collagen molecules in vivo.

[0039] “Crosslinkable peptides”: peptides containing a fragment of thecollagen sequence including at least one crosslinkable site.

[0040] Test kit: A combination of reagents and instructions for use inconducting an assay.

[0041] “Essentially derived” (about structures): Structures with similarantigenicity, i.e. with an ability, above the level of a non-relatedpeptide, to inhibit the binding of any of the mentioned syntheticpeptides to an antibody immunoreactive with said synthetic peptide.

[0042] It is contemplated that the method may also be used for assayingcollagen fragments in animal body fluids, e.g. for determination of thecollagen metabolism. It also can be used during clinical testing of newdrugs to assess the impact of these drugs on collagen metabolism.

[0043] More specifically, the present invention relates to methods forassaying collagen fragments by the use of synthetic peptidescorresponding to the above-mentioned sequences of collagen. Generally,these synthetic peptides will have fewer amino acid residues than theentire collagen molecule, often they will have fewer than 10 aminoacids. Also, the synthetic peptides, corresponding to molecules presentin body fluids, e.g. urine, will have potential sites for cross-linking,preferably lysine or hydroxylysine, incorporated in the structure.

[0044] The present invention comprises the determination of collagenfragments by the use of antibodies which are immunoreactive with theabove-mentioned synthetic peptides, said peptides each having a sequencederived from collagen fragments having crosslinkable sites.

[0045] The invention also includes cell lines (e.g. hybridomas) thatproduce monoclonal antibodies immunoreactive with the above-mentionedsynthetic peptides. The invention further includes monoclonal antibodiesproduced by the fused cell hybrids, and those antibodies (as well asbinding fragments thereof, e.g. Fab) coupled to a detectable marker.Examples of detectable markers include, but are not limited to, enzymes,chromophores, fluorophores, coenzymes, enzyme inhibitors,chemiluminescent materials, paramagnetic metals, spin labels andradioisotopes.

[0046] The methods of the invention involve quantitating in a body fluidthe concentration of particular collagen fragments derived from collagendegradation. In a representative assay, collagen fragments in thepatient's body fluid and a synthetic peptide immobilized on a solidsurface are contacted with an immunological binding partner which isimmunoreactive with the synthetic peptide. Suitable body fluids are e.g.human urine, blood, serum, plasma and synovial fluid. It is contemplatedthat the method may also be used e.g. on saliva and sweat. The bodyfluid may be used as it is, or it may be purified prior to thecontacting step. This purification step may be accomplished using anumber of standard procedures, including, but not limited to, cartridgeadsorption and elution, molecular sieve chromatography, dialysis, ionexchange, alumina chromatography, hydroxyapatite chromatography, andcombinations thereof.

[0047] The present invention is based on the discovery of simpleprocedures for quantitation of collagen fragments in body fluids. In arepresentative procedure, synthetic peptides containing potential sitesfor cross-linking, are used for the raising of antibodies andsubsequently incorporated in the assay for quantitation of collagenfragments generated in vivo by collagen degradation.

[0048] The invention also includes test kits useful for quantitating ina body fluid the amount of collagen fragment derived from thedegradation of collagen. The kits comprise at least one immunologicalbinding partner, e.g. a monoclonal or polyclonal antibody specific for apeptide derived from the degradation of collagen. If desired, theimmunological binding partner of the test kit may be coupled todetectable markers such as the ones described above.

[0049] The invention is described in more detail below. Reference ismade to the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIGS. 1, 2 and 3 are typical standard curves for the α1(I)C1immunoassay (FIG. 1), the a1(I)N1 immunoassay (FIG. 2) and the α2(I)N1immunoassay (FIG. 3) to be described in more detail in the examples;

[0051]FIG. 4 shows the correlation between total pyridinolin (HPLC) andthe α1(I)C1 immunoassay,

[0052]FIG. 5 shows the correlation between total pyridinolin (HPLC) andthe α1(I)N1 immunoassay,

[0053]FIG. 6 shows the individual values of the measurement of α1(I)C1peptide in urine samples from normal, age-matched women, and

[0054]FIG. 7 shows the individual values of the measurement of urinesamples from women on a hormone replacement therapy and placebo in theα1(I)C1 immunoassay.

DETAILED DESCRIPTION AND BEST MODE OF CARRYING OUT THE INVENTION

[0055] In a preferred embodiment of the method according to theinvention, the assaying of type I, II and III collagen fragments inurine is performed by an inhibition ELISA (enzyme linked immunosorbentassay) by metering off a sample of urine and contacting the sample witha synthetic peptide having a sequence derived from collagen and with anantibody, which is immunoreactive with the synthetic peptide. Thesynthetic peptide is immobilized on a solid support. The antibody israised against the synthetic peptide.

[0056] The combined reagents and sample are incubated, and aperoxidase-conjugated (revealing) antibody is added. After anotherincubation, a peroxidase substrate solution is added. Following shortfinal incubation, the enzyme reaction is stopped, and the absorbance ismeasured at 450 nm and compared with a standard curve obtained withstandard solutions by the same procedure.

[0057] Synthetic peptides are used for the preparation of standards. Theconcentration of synthetic peptide in a stock solution of the relevantsynthetic peptide is determined by quantitative amino aciddetermination. A two-fold dilution of the stock solution is prepared andsubsequently used for the construction of the standard curve in theinhibition ELISA.

[0058] Preparation of Synthetic Peptides

[0059] The preparation of synthetic peptides may be performed accordingto procedures well known in the art, e.g. by solid-phase peptidesynthesis techniques commonly described as “Merrifield synthesis”. Alsoclassical solution phase techniques may be used. Sequences of interestinclude potential sites for cross-linking (see for example Kühn, K., inImmunochemistry of the Extracellular Matrix 1:1-29 (1982), Eyre, D. R.,Ann. Rev. Biochem. 53:717-48 (1984), or U.S. Pat. No. 5,140,103).Examples of such peptides sequences are given in table 1 below.

[0060] Regarding the synthetic peptides, it is possible to omit (or add)one or more amino acid residues from (or to) the crosslinkable sitesequences without substantial loss of the ability to (a) raiseantibodies recognizing the corresponding native collagen fragment or (b)inhibit the bindings of such antibodies to the native fragment. It ispossible to use longer collagen fragments and/or chimeric peptides toraise the antibodies and, in principle, it is not necessary to use thesame peptide as the immunogen and the competitor in the assay.

Table 1

[0061] Examples of Amino acid sequences with potential sites forcross-linking in various types of collagen to be used as a basis forsynthetic peptides according to the present invention

[0062] Collagen type I

[0063] Potential sites in telopeptides: N C

[0064] α1 (I) N-term. Asp-Glu-Lys-Ser-Thr-Gly-Gly (α1(I)N1)

[0065] α1 (I) C-term. Glu-Lys-Ala-His-Asp-Gly-Gly-Arg (α1(I)C1)

[0066] α2 (I) N-term. Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (α2(I)N1)

[0067] α2 (I) C-term. no potential sites

[0068] Potential sites in helix:

[0069] α1 (I) (near N) Gly-Met-Lys-Gly-His-Arg

[0070] α1 (I) (near C) Gly-Ile-Lys-Gly-His-Arg

[0071] α2 (I) (near N) Gly-Phe-Lys-Gly-Ile-Arg

[0072] α2 (I) (near C) Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn

[0073] Collagen Type II

[0074] Potential sites in telopeptides: N C

[0075] α1 (II) N-term. Pro-Gly-Pro-Lys-Gly-Glu Gly-Gln-Lys-Gly-Glu-ProGly-Asp-Ile-Lys-Asp-Ile-Val

[0076] α1 (II) C-term. Glu-Lys-Gly-Pro-Asp

[0077] Potential sites in helix:

[0078] α1 (II) (near N) Gly-Val-Lys Pro-Gly-Val-Lys-Gly

[0079] Collagen Type III

[0080] Potential sites in telopeptides: N C

[0081] α1 (III) N-term. Asp-Val-Lys-Ser-Gly-Val

[0082] α1 (III) C-term. Glu-Lys-Ala-Gly-Gly-Phe-Ala

[0083] Potential sites in helix:

[0084] α1 (III) (near N) Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg

[0085] α1 (III) (near C) Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg

[0086] Preparation of Antibodies

[0087] The methods for preparation of both monoclonal and polyclonalantibodies are well known in the art. For example, see Campbell, A. M.,Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13(1986). It is possible to produce antibodies to synthetic peptides byimmunization. However, because of the relatively small molecular weightof these compounds it is preferred that the hapten be conjugated to acarrier molecule. Suitable carrier molecules include, but are notlimited to, bovine serum albumin, thyroglobulin, ovalbumin, tetanustoxoid, and keyhole limpet hemocyanin. The preferred carrier is bovineserum albumin. To present the hapten in its most immunogenic form to theantibody producing cells of the immunized animal a number of alternativecoupling protocols can be used. Suitable procedures include, but are notlimited to, glutaraldehyde, carbodiimide, and periodate. Preferredbinding agents are glutaraldehyde and carbodiimide.

[0088] The preparation of antibodies is carried out by conventionaltechniques including immunization with collagen fragments or syntheticpeptides conjugated to a carrier. To improve the immunogenicity it ispreferred that the immunogen be mixed with an adjuvant before injection.Examples of adjuvants include, but are not limited to, aluminumhydroxide, Freund's adjuvant, and immune-stimulating complexes (ISCOMs).ISCOMs can be made according to the method described by Morein, B. etal., Nature 308:457-460 (1984).

[0089] Either monoclonal or polyclonal antibodies to the hapten carriermolecule can be produced. For the production of monoclonal antibodies itis preferred that mice are immunized. Spleen cells from the immunizedmouse are harvested, homogenized, and thereafter fused with cancer cellsin the presence of polyethylene glycol to produce a cell hybrid whichproduces monoclonal antibodies specific for peptide fragments derivedfrom collagen. Suitable cancer cells include, but are not limited to,myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed descriptionsof the production of monoclonal antibodies are provided in Goding, J.W., in Monoclonal Antibodies: Principles and Practice, (1986). Apreferred preliminary screening protocol comprises the use of syntheticpeptides conjugated to a carrier and coated onto the solid surface of amicrotitre plate.

[0090] For the preparation of polyclonal antibodies, which are reactivewith peptide fragments derived from collagen, different animal speciescan be immunized. Suitable species include, but are not limited to,chicken, rabbit and goat. Chicken and rabbit are preferred.

[0091] Antibody fragments are prepared by methods known in the art (seeE. Ishikawa, J. of Immunoassay 3:209-327, 1983).

[0092] Conduct of Immunoassays

[0093] Accordingly, by utilization of an immunoassay with the antibodiesprepared as above it is possible to assay a biological fluid samplewithout prior fractionation or hydrolysis. The specificity for thedesired collagen in the biological fluid is supplied by the antibody incombination with the use of a synthetic peptide (against which theantibody was raised or in any event with which the antibody isimmunochemically reactive) in the assay construction.

[0094] The immunoassays themselves are conducted using any procedureselected from the variety of standard assay protocols generally known inthe art. As it is generally understood, the assay is constructed so asto rely on the interaction between the specific immunological bindingpartner and the desired analyte for specificity and to utilize somemeans to detect the complex formed by the analyte and the immunologicalbinding partner. The immunological binding partner may be complexed to asolid support and used as a capture immunological binding partner forthe analyte. This protocol may be run in a direct form, wherein theformation of analyte/immunological binding partner complex is detected,e.g. by a fluorescent, radioactive or enzymatic label, or it may be runin a competitive format wherein a labelled standard competes with theanalyte for the immunological binding partner. The format may also beconstructed as an agglutination assay or the complex may be precipitatedby addition of a suitable precipitant to the reaction mixture. Thespecific design of the immunoassay protocol is open to a wide variety ofchoice, and the number of clinical assay devices and protocols availablein the art is multitudinous. For a variety of such protocols, see U.S.Pat. No. 5,001,225.

[0095] The antibodies and revealing reagents for the conduct of animmunoassay using standard detection protocols, for example radioisotopelabelling, fluorescent labelling or ELISA, either in a direct orcompetitive format, may conveniently be supplied as kits which includethe necessary components and instructions for the assay. In oneembodiment of the invention such a kit includes a microtiter platecoated with a relevant synthetic peptide, standard solutions forpreparation of standard curve, a urine control for quality testing ofthe analytical run, rabbit antibodies reactive with the above-mentionedsynthetic peptide, anti-rabbit immunoglobulins conjugated to peroxidase,a substrate solution, a stopping solution, a washing buffer and aninstruction manual.

[0096] Since immunoassays can be constructed using antibodies andspecific synthetic peptides, the ratios of the corresponding collagenfragment sequences in an appropriate biological fluid can be determinedas well as their individual levels and their total. Thus, the assay canbe designed to include antibodies which will result in determination ofseveral native peptide sequences or determination of a single peptidesequence, or any desired combination thereof.

[0097] In addition to the use of the herein specified peptides asindicators of bone resorption, bone metabolic balance is advantageouslydetermined by the substantially simultaneous determination of a markerof the formation of bone in the same or other appropriate biologicalfluid from the same individual. “Substantially simultaneous” means thesame day, preferably within 4 hours. For example such markers includeosteocalcin (also known as bone GLA protein of BGP), procollagen type I,bone alkaline phosphatase and total alkaline phosphatase. Suitablemethods for the determination of these markers can be found, forexample, in Delmas, P. D. , et al. , J. Bone Min. Res. 1:333-337 (1986).

[0098] The assay of the present invention which provides an index todetermination of the metabolic status of tissues, which generatecollagen-derived peptides when degradation occurs, are useful in avariety of contexts. First, when considering the degradation of type Icollagen, the assays are methods to assess an abnormal condition of asubject by indicating, for example, excessive bone resorption. This mayshow the presence of an osteoporotic condition or the metastaticprogress of a malignancy. Other conditions characterized by excessivebone resorption include Paget's disease and hyperparathyroidism.Likewise, a number of other disease states involving connective tissuemay be monitored by determination of the degradation of collagen.Examples are collagen type II degradation associated with rheumatoidarthritis and osteoarthritis and collagen type III degradation invasculitis syndrome. Since the condition of the subject can be monitoredcontinuously, application of these assays can also be used to monitorthe progress of therapy administered to treat these or other conditions.Further, the assays can be used as a measure of toxicity, since theadministration of toxic substances often results in tissue degradation.

[0099] Thus the assays may be applied in any situation wherein themetabolic condition of collagen tissues can be used as an index of thecondition, treatment, or effect of substances directly administered tothe subject or to which the subject is exposed in the environment.

[0100] The following examples are intended to illustrate, but not tolimit the invention.

EXAMPLE 1

[0101] Immunoassays for Specific Peptide Sequences in Urine

[0102] Three peptides (α1(I)C1, α1(I)N1, and α2(I)N1) (see Table 1, p.13) prepared by solid-phase techniques are used for the preparation ofimmunogens. For immunization, the peptides are covalently attached tobovine serum albumin using glutaraldehyde reagents and methods wellknown in the art. Both monoclonal and polyclonal antibodies are raisedagainst the peptides. For production of monoclonal antibodies, Balb/cmice are immunized with peptide-BSA conjugates, and hybridoma cell linesare prepared using standard techniques after fusion of cells from thespleen or lymph nodes with Ag8 myeloma cells. Polyclonal antibodies areraised in rabbits and chicken. Screening of both antisera and hybridomacell media were performed by ELISA using microtiter plates coated withthe appropriate peptide-gelatin conjugate prepared using carbodiimidereagents and methods well known in the art.

[0103] Assays for three of the peptide sequences (α1(I)C1, α1(I)N1, andα2(I)N1) in urine are performed by an inhibition ELISA as follows:

[0104] Urine samples (10 or 25 l) possibly containing collagen fragmentsor solutions containing 0.05-15 g peptide/ml as reference standards,respectively, are added to 75 l of immunological binding partners forthe peptides diluted 1:5,000-1:20,000 in phosphate buffered salinecontaining 0.1% Tween-20 detergent (PBS-T) and including 0.1% (w/v) ofBSA. Each sample is prepared in duplicate in flat-bottomed, 96-wellmicrotiter plates previously coated with gelatine conjugate containingthe appropriate peptide. After 60 minutes, the plates are washed withPBS-T (3 times) and the bound antibodies are detected by standardtechniques with a horse radish peroxidase labelled antibody preparedagainst the species of the primary antibody. Peroxidase substrate isadded and the color development is measured at 450 nm in an automatedmicrotiter plate reader after stopping the enzyme reaction using 1 MH3PO4. Samples containing the analyte decrease the binding of primaryantibody to the immunobilized peptide in the plate and thus have areduced color concentration. The amount of analyte in the sample isquantified with reference to previously established curves fromstandards included on each plate computed using log-lin plots. FIGS. 1,2 and 3 show typical standard curves for the α1(I)C1 immunoassay (FIG.1), α1(I)N1 immunoassay (FIG. 2), and α2(I)N1 immunoassay (FIG. 3).

EXAMPLE 2

[0105] Correlation to Pyridinolin Determination on HPLC

[0106] On a number of unselected urine samples the concentration oftotal pyridinolin (HPLC method, see for example Uebelhart, D., Bone andMineral 8:87-96 (1990)) was measured. Values obtained in this HPLCsystem were correlated to the values obtained in two immunoassays(α1(I)C1 peptide and α1(I)N1 peptide).

[0107]FIG. 4 shows the correlation between total pyridinolin (HPLC) andα1(I)C1 immunoassay (n=59). The correlation calculated by linearregression analysis is r=0.80.

[0108]FIG. 5 shows the correlation between total pyridinolin (HPLC) andα1(I)N1 immunoassay (n=36). The correlation calculated by linearregression analysis is r=0.95.

EXAMPLE 3

[0109] Clinical Results

[0110] The immunoassay procedure (using the α1(I)C1 peptide) set forthin Example 1 was applied to urine samples from different individuals,and the amount of analyte was quantitated. The values obtained wererelated to the level of urinary creatinine in the urine sample as iscommonly done for urine assays. The values obtained for normal,age-matched women (premenopausal and postmenopausal) are shown in Table2. TABLE 2 Measurement of α1(I)C1 peptide in urine samples from normal,age-matched women (premenopausal and postmenopausal) Group α1(T)C1peptide (g/mol creatinine) Premenopausal (n = 104) 0.263 ± 0.143Postmenopausal (n = 180) 0.426 ± 0.190

[0111] For individual values see FIG. 6.

[0112] The difference between the premenopausal and the post-menopausalvalues is highly significant (P<0.0001).

[0113] The z-score of a quantitative test procedure shows the ability ofthe procedure to distinguish between two populations. Table 3 belowshows the z-score of the IC1 immunoassay and the state of the artmeasurement of pyridinolin on HPLC when applied to the same set of urinesamples from normal, age-matched premenopausal (n=104) andpostmenopausal women (n=180). TABLE 3 Z-scores obtained for twodifferent test procedures (total pyridinolin (HPLC) and α1(I)C1immunoassay) when applied to urine samples from normal, age-matchedwomen (premenopausal and postmenopausal) Test procedure Z-score α1(I)C1immunoassay 1.14 Total pyridinolin (HPLC) 1.25

[0114] As can be seen from Table 3, the abilities of the two methods todistinguish between patient populations are approximately alike.

[0115] For an assay to be used as an index of bone resorption it is veryimportant to be able to measure the impact of a hormone replacementtherapy (HRT). Table 4 shows the results from the α1(I)C1 immunoassay insuch a study. TABLE 4 Measurement of urine samples from women on ahormone replacement therapy and placebo in the α1(I)C1 immunoassay.Values given as percent of t = 0 α1(I)C1 peptide (μg/mol creatinine)Group t = 0 t = 12 months HRT (n = 92) 0.457 ± 0.176 0.176 ± 0.103Placebo (n = 45) 0.459 ± 0.209 0.402 ± 0.187

[0116] For individual values see FIG. 7.

[0117] A highly significant drop in the group receiving HRT is seenafter 12 months (P<0.001).

[0118] All cited patents, patent applications and literature areincorporated by reference in their entirety. In case of conflict,however, the present disclosure controls.

[0119] The invention has been described above by reference to specificembodiments It will be apparent to those skilled in the art, however,that many additions, deletions and modifications are possible withoutdeparting from the spirit of the invention as claimed below.

[0120] Collagen alpha 1(I) chain precursor—Human (fragments) Homosapiens (man) Number of residues=1341        5        10        15        20        25        30    1 M F S FV D L R L L L L L A A T A L L T H G Q E E G Q V E G   31 Q D E D I P P IT C V Q N G L R Y H D R D V W K P E P C Q I   61 C V C D N G K V L C D DV I C D E T K N C P G A E V P E G E   91 C C P V C P D G S E S P T D Q ET T G V E G P K G D T G P R  121 G P R G P A G P P G R D G I P G Q P G LP G P P G P P G P P  151 G P P G L G G N F A P Q L S Y G Y D E K S T G GI S V P G P  181 M(G P S G P R G L P G P P G A P G P Z G F Z G P P G Z PG Z  211 P G A S G P)M(G P R G P P G P P G K B G B B G Z A G K P G R 241 P G Z R G P P G P Z G A R G L P G T A G L P)G M K G H R(G F  271 SG L B G A K G B A G P A G P K G Z P G S P G Z B G A P G Z)  301 M\G P PG P K G N S G E P G A P G S K G D T G A K G E P G P  331 V G V Q G P P GP A G E E G K R G A R G E P G P T G L P G P  361 P G E R G G P G S R G FP G A D G V A G P K G P A G E R G S  391 P G P A G P K G S P G E A G R PG E A G L P G A K G L T G S  421 P G S P G P D G K T G P P G P A G Q D GR P G P P G P P G A  451 R G Q A G V M G F P G P K G A A G E P G K A G ER G V P G P  481 P G A V G P A G K D G E A G A Q G P P G P A G P A G E RG E  511 Q G P A G S P G F Q G L P G P A G P P G E A G K P G E Q G V 541 P G D L G A P G P S G A R G E R G F P G E R G V Q G P P G P  571 AG P R G A N G A P G N D G A K G D A G A P G A P G S Q G A  601 P G L Q GM P G E R G A A G L P G P K G D R G D A G P K G A  631 D G S P G K D G VR G L T G P I G P P G P A G A P G D K G E  661 S G P S G P A G P T G A RG A P G D R G E P G P P G P A G F  691 A G P P G A D G Q P G A K G E P GD A G A K G D A G P P G P  721 A G P A G P P G P I G N V G A P G A K G AR G S A G P P G A  751 T G F P G A A G R V G P P G P S G N A G P P G P PG P A G K  781 E G G K G P R G E T G P A G R P G E V G P P G P P G P A GE  811 K G S P G A D G P A G A P G T P G P Q G I A G Q R G V V G L  841P G Q R G E R G F P G L P G P S G E P G K Q G P S G A S G E  871 R G P PG P M G P P G L A G P P G E S G R E G A P G A E G S  901 P G R D G S P GA K G D R G E T G P A G P P G A X G A X G A  931 P G P V G P A G K S G DR G E T G P A G P A G P V G P A G A  961 R G P A G P Q G P R G D K G E TG E Q G D R G I K G H R G F  991 S G L Q G P P G P P G S P G E Q G P S GA S G P A G P R G P 1021 P G S A G A P G K D G L N G L P G P I G P P G PR G R T G D 1051 A G P V G P P G P P G P P G P P G P P S A G F D F S F LP Q 1081 P P Q E K A H D G G R Y Y R A D D A N V V R D R D L E V D T1111 T L K S L S Q Q I E N I R S P E G X R K N P A R T C R D L K 1141 MC H S D W K S G E Y W I D P N Q G C N L D A I K V F C N M 1171 E T G E TC V Y P T Q P S V A Q K N W Y I S K N P K D K R H 1201 V W F G E S M T DG F Q F E Y G G Q G S D P A D V A I Q L T 1231 F L R L M S T E A S Q N IT Y H C K N S V A Y M D Q Q T G N 1261 L K K A L L L X G S N E I E I R AE G N S R F T Y S V T V D 1291 G C T S H T G A W G K T V I E Y K T T K SS R L P I I D V A 1321 P L D V G A P D Q E F G F D V G P V C F L

[0121] Sequence of collagen alpha 1(I) chain precursor prepared from:

[0122] Chu, M. L., de Wet, W., Bernard, M., Ding, J. F., Morabito, M.,Myers, J., Williams, C., and Ramirez, F., Nature 310, 337-340, 1984(Human, sequence of residues 1-181 translated from the DNA sequence)

[0123] Click, E. M., and Bornstein, P., Biochemistry 9, 4699-4706, 1970(Human skin, CNBr0-1, CNBr2, CNBr4, CNBr5, partial sequence of residues162-301)

[0124] Morgan, P. H., Jacobs, H. G., Segrest, J. P., and Cunningham, L.W., J. Biol. Chem. 245, 5042-5048, 1970 (Human skin, sequence ofresidues 263-268)

[0125] Bernard, M. P., Chu, M. L., Myers, J. C., Ramirez, F.,Eikenberry, E. F., and Prockop, D. J., Biochemistry 22, 5213-5223, 1983(Sequence of residues 302-1341 translated from the mRNA sequence)

[0126] Procollagen alpha 2(I) chain precursor Homo sapiens (man) Numberof residues=1366        5        10        15        20        25        30    1 M L S FV D T R T L L L L A V T L C L A T C Q S L Q E E T V   31 R K G P A G D RG P R G E R G P P G P P G R D G E D G P T G   61 P P G P P G P P G P P GL G G N F A A Q Y D G K G V G L G P   91 G P M G L M G P R G P P G A A GA P G P Q G F Q G P A G E P  121 G E P G Q T G P A G A R G P A G P P G KA G E D G H P G K P  151 G R P G E R G V V G P Q G A R G F P G T P G L PG F K G I R  181 G H N G L D G L K G Q P G A P G V K G E P G A P G E N GT P  211 G Q T G A R G L P G E R G R V G A P G P A G A R G S D G S V 241 G P V G P A G P N G S A G P P G F P G A P G P K G E I G A V  271 GN A G P T G P A G P R G E V G L P G L S G P V G P P G N P  301 G A N G LT G A K G A A G L P G V A G A P G L P G P R G I P  331 G P P G A A G T TG A R G L V G E P G P A G S K G E S G N K  361 G E P G S A G P Q G P P GP S G E E G K R G P N G E A G S A  391 G P P G P P G L R G S P G S R G LP G A D G R A G V M G P P  421 G S R G A S G P A G V R G P N G D A G R PG E P G L M G P R  451 G L P G S P G N I G P A G K E G P V G L P G I D GR P G P I  481 G P V G A R G E P G N I G F P G P K G P T G D P G K N G DK  511 G H A G L A G A R G A P G P D G N N G A Q G P P G P Q G V Q  541G G K G E Q G P A G P P G F Q G L P G P S G P A G E V G K P  571 G E R GL H G E F G L P G P A G P R G E R G P P G E S G A A  601 G P T G P I G SR G P S G P P G P D G N K G E P G V V G A V  631 G T A G P S G P S G L PG E R G A A G I P G G K G E K G E P  661 G L R G E I G N P G R D G A R GA H G A V G A P G P A G A T  691 G D R G E A G A A G P A G P A G P R G SP G E R G E V G P A  721 G P N G F A G P A G A A G Q P G A K G E R G G KG P K G E N  751 G V V G P T G P V G A A G P A G P N G P P G P A G S R GD G  781 G P P G M T G F P G A A G R T G P P G P S G I S G P P G P P 811 G P A G K E G L R G P R G D Q G P V G R T G E V G A V G P P  841 GF A G E K G P S G E A G T A G P P G T P G P Q G L L G A P  871 G I L G LP G S R G E R G L P G V A G A V G E P G P L G I A  901 G P P G A R G P PG A V G S P G V N G A P G E A G R D G N P  931 G N D G P P G R D G Q P GH K G E R G Y P G N I G P V G A A  961 G A P G P H G P V G P A G K H G NR G E T G P S G P V G P A  991 G A V G P R G P S G P Q G I R G D K G E PG E K G P R G L P 1021 G F K G H N G L Q G L P G I A G H H G D Q G A P GS V G P A 1051 G P R G P A G P S G P A G K D G R T G H P G T V G P A G IR 1081 G P Q G H Q G P A G P P G P P G P P G P P G V S G G G Y D F 1111G Y D G D F Y R A D Q P R S A P S L R P K D Y E V D A T L K 1141 S L N NQ I E T L L T P E G S R K N P A R T C R D L R L S H 1171 P E W S S G Y YW I D P N Q G C T M E A I K V Y C D F P T G 1201 E T C I R A Q P E N I PA K N W Y R S S K D K K H V W L G E 1231 T I N A G S Q F E Y N V E G V TS K E M A T Q L A F M R L L 1261 A N Y A S Q N I T Y H C K N S I A Y M DE E T G N L K K A V 1291 I L Q G S N D V E L V A E G N S R F T Y T V L VD G C S K K 1321 T N E W G K T I I E Y K T N K P S R L P F L D I A P L DI G 1351 G A D H E F F V D I G P V C F K

[0127] Sequence of procollagen alpha 2(I) chain precursor prepared from:

[0128] de Wet, W., Bernard, M., Benson-Chanda, V., Chu, M. L., Dickson,L., Weil, D., and Ramirez, F., J. Biol. Chem. 262, 16032-16036, 1987(Sequence translated from the mRNA sequence) Title: Organization of thehuman pro-alpha-2(I) collagen gene.

[0129] Collagen alpha 1(II) chain precursor Homo sapiens (man) Number ofresidues=1418    1 MIRLGAPQSL VLLTLLVAAV LRCQGQDVRQ PGPKGQKGEPGDIKDIVGPK   51 GPPGPQGPAG EQGPRGDRGD KGEKGAPGPR GRDGEPGTLG NPGPPGPPGP 101 PGPPGLGGNF AAQMAGGFDE KAGGAQLGVM QGPMGPMGPR GPPGPAGAPG  151PQGFQGNPGE PGEPGVSGPM GPRGPPGPPG KPGDDGEAGK PGKAGERGPP  201 GPQGARGFPGTPGLPGVKGH RGYPGLDGAK GEAGAPGVKG ESGSPGENGS  251 PGPMGPRGLP GERGRTGPAGAAGARGNDGQ PGPAGPPGPV GPAGGPGFPG  301 APGAKGEAGP TGARGPEGAQ GPRGEPGTPGSPGPAGASGN PGTDGIPGAK  351 GSAGAPGIAG APGFPGPRGP PDPQGATGPL GPKGQTGKPGIAGFKGEQGP  401 KGEPGPAGPQ GAPGPAGEEG KRGARGEPGG VGPIGPPGER GAPGNRGFPG 451 QDGLAGPKGA PGERGPSGLA GPKGANGDPG RPGEPGLPGA RGLTGRPGDA  501GEQGKVGPSG APGEDGRPGP PGPQGARGQP GVMGFPGPKG ANGEPGKAGE  551 KGLPGAPGLRGLPGKDGETG AEGPPGPAGP AGERGEQGAP GPSGFQGLPG  601 PPGPPGEGGK PGDQGVPGEAGAPGLVGPRG ERGFPGERGS PGAQGLQGPR  651 GLPGTPGTDG PKGASGPAGP PGAQGPPGLQGMPGERGAAG IAGPKGDRGD  701 VGEKGPEGAP GKDGGRGLTG PIGPPGPAGA NGEKGEVGPPGPAGSAGARG  751 APGERGETGP PGTSGIAGPP GADGQPGAKG EQGEAGQKGD AGAPGPQGPS 801 GAPGPQGPTG VTGPKGARGA QGPPGATGFP GAAGRVGPPG SNGNPGPPGP  851PGPSGKDGPK GARGDSGPPG RAGEPGLQGP AGPPGEKGEP GDDGPSGAEG  901 PPGPQGLAGQRGIVGLPGQR GERGFPGLPG PSGEPGQQGA PGASGDRGPP  951 GPVGPPGLTG PAGEPGREGSPGADGPPGRD GAAGVKGDRG ETGAVGAPGA 1001 PGPPGSPGPA GPTGKQGDRG EAGAQGPMGPSGPAGARGIQ GPQGPRGDKG 1051 EAGEPGERGL KGHRGFTGLQ GLPGPPGPSG DQGASGPAGPSGPRGPPGPV 1101 GPSGKDGANG IPGPIGPPGP RGRSGETGPA GPPGNPGPPG PPGPPGPGID1151 MSAFAGLGPR EKGPDPLQYM RADQAAGGLR QHDAEVDATL KSLNNQIESI 1201RSPEGSRKNP ARTCRDLKLC HPEWKSGDYW IDPNQGCTLD AMKVFCNMET 1251 GETCVYPNPANVPKKNWWSS KSKEKKHIWF GETINGGFHF SYGDDNLAPN 1301 TANVQMTFLR LLSTEGSQNITHYCKNSIAY LDEAAGNLKK ALLIQGSNDV 1351 EIRAEGNSRF TYTALKDGCT KHTGKWGKTVIEYRSQKTSR LPIIDIAPMD 1401 IGGPEQEFGV DIGVCFL

[0130] Sequence of collagen alpha 1(II) chain precursor prepared from:

[0131] Su, M. W., Lee, B., Ramirez, F., Machado, M., and Horton, W.Nucleic Acids Res 17, 9473, 1989 Nucleotide sequence of the full lengthcDNA encoding for human type II procollagen.

[0132] Baldwin, C. T., Reginato, A. M., Smith, C., Jimenez, S. A., andProckop, D. J., Biochem. J. 262, 521-528, 1989 Structure of cDNA clonescoding for human type II procollagen The alpha-1(II) chain is moresimilar to the alpha-1(I) chain than two other alpha chains of fibrillarcollagens.

[0133] Ala-Kokko, L., Baldwin, C. T., Moskowitz, R. W., and Prockop, D.J. Proc. Natl. Acad. Sci. U.S.A. 87, 6565-6568, 1990 Single basemutation in the type II procollagen gene (COL2A1) as a cause of primaryosteoarthritis associated with a mild chondrodysplasia.

[0134] Ramirez, F. submitted to the EMBL Data Library, December 1988Reference number: S04892

[0135] Vikkula, M., and Peltonen, L. FEBS Lett. 250, 171-174, 1989Structural analyses of the polymorphic area in type II collagen gene.

[0136] Collagen alpha 1(III) chain precursor Homo sapiens (man) Numberof residues=1078        5        10        15        20        25        30    1 \Q N YS P Q Y D S Y D V K S G G V A V G G L A G Y P G P A G   31 P P G P P G PP G T S G H P G S P G S P G Y Q G P P G E P G   61 Q A G P S G P P G P PG A I G P S G P A G K D G E S G R P C   91 R P G D R G L P G P P G I K GP A G I P G F P G M K G H R G  121 F D G R N G E K G E T G A P G L K G EN G L P G E N G A P G  151 P M G P R G A P G E R G R P G L P G A A G A RG N D G A R G  181 S D G Q P G P P G P P G T A G F P G S P G A K G E F GP A G  211 S P G S N G A P G Q R G E P G P Q G H A G A Q G P P G P P G 241 I N G S P G G K G E M G P A G I P G A P G L M G A R G P P G  271 PA G A N G A P G L R G G A G E P G K N G A K G E P G P R G  301 E R G E AG I P G V P G A K G E D G K D G S P G D P G A N G  331 L P G A A G E R GA L G S R G P A G P N G I P G E K G P A G  361 E R G A P G P A G P R G AA G E P G R D G V P G G P G M R G  391 M P G S P G G P G S D G K P G P PG S Q G E S G R P G P P G  421 P S G P R G Q P G V M G F P G P K G N D GA P G K N G E R G  451 G P G G P G P Q G P P G K N G E Y G P Q G P P G PT G P G P  481 D K G D T G P R G P Q G L Q G L P G T G G P P G E N G K PG  511 E P G P K G E A G A P G A P G G K G D A G A P G E R G P P G  541L A G A P G L R G G A G P P G P E G G K G A A G P P G P P G  571 A A G TP G L Q G M P G E R G G L G S P G P K G D K G E P G  601 G P G A D G V PG K D G P R G P T G P I G P P G P A G Q P G  631 D K G E G G A P G L P GI A G P R G S P G E R G E T G P P G  661 P A G F P G A P G Q N G E P G GK G E R G A P G E K G E G G  691 P P G V A G P P G G S G P A G P P G P QG V K G E R G S P G  721 G P G A A G F P G A R G L P G P P G S N G N P GP P G P S G  751 S P G K D G P P G P A G N T G A P G S P G V S G P K G DA G  781 Q P G E K G S P G A Q G P P G A P G P L G I A G I T G A R G 811 L A G P P G M P G P R G S P G P Q G V K G E S G K P G A N G  841 LS G E R G P P G P Q G L P G L A G T A G E P G R D G N P G  871 S D G L PG R D G S P G G K G D R G E N G S P G A P G A P G  901 H P G P P G P V GP A G K S G D R G E S G P A G P A G A P G  931 P A G S R G A P G P Q G PR G D K G E T G E R G A A G I K G  961 H R G F P G N P G A P G S P G P AG Q Q G A I G S P G P A G  991 P R G P V G P S G P P G K D G T S G H P GP I G P P G P R G 1021 N R G E R G S E G S P G H P G Q S G P P G P P G AP G P C C 1051 G G V G A P A I A G I G A E K A G G F A P Y Y G D E P M\

[0137] Sequence of collagen alpha 1(III) chain precursor prepared from:

[0138] Janeczko, R. A., and Ramirez, F., Nucleic Acids Res. 17, 6742,1989 Nucleotide and amino acid sequences of the entire human alpha-1(III) collagen.

1 21 7 amino acids amino acid linear peptide N-terminal Homo sapiensCOLLAGEN TYPE I-alpha 1 -N term 1 Asp Glu Lys Ser Thr Gly Gly 1 5 8amino acids amino acid linear peptide C-terminal Homo sapiens COLLAGENTYPE I -ALPHA 1- c TERMINAL 2 Glu Lys Ala His Asp Gly Gly Arg 1 5 8amino acids amino acid linear peptide N-terminal Homo sapiens COLLAGENTYPE I - ALPHA 2- N TERMINAL 3 Gln Tyr Asp Gly Lys Gly Val Gly 1 5 6amino acids amino acid linear peptide internal Homo sapiens COLLAGENTYPE I -ALPHA 1- NEAR N 4 Gly Met Lys Gly His Arg 1 5 6 amino acidsamino acid linear peptide C-terminal Homo sapiens COLLAGEN TYPE I -ALPHA1 NEAR c 5 Gly Ile Lys Gly His Arg 1 5 6 amino acids amino acid linearpeptide internal Homo sapiens COLLAGEN TYPE I- ALPHA 2- NEAR N 6 Gly PheLys Gly Ile Arg 1 5 9 amino acids amino acid linear peptide internalHomo sapiens COLLAGEN TYPE I - ALPHA 2 - NEAR C 7 Gly Leu Pro Gly LeuLys Gly His Asn 1 5 6 amino acids amino acid linear peptide N-terminalHomo sapiens COLLAGEN TYPE II- ALPHA 1- N TERM 8 Pro Gly Pro Lys Gly Glu1 5 6 amino acids amino acid linear peptide N-terminal Homo sapiensCOLLAGEN TYPE II- ALPHA 1 N2 9 Gly Gln Lys Gly Glu Pro 1 5 7 amino acidsamino acid linear peptide N-terminal Homo sapiens COLLAGEN TYPE II -ALPHA 1- N3 10 Gly Asp Ile Lys Asp Ile Val 1 5 5 amino acids amino acidlinear peptide C-terminal Homo sapiens COLLAGEN TYPE II - ALPHA 1 -CTERM 11 Glu Lys Gly Pro Asp 1 5 3 amino acids amino acid linear peptideinternal Homo sapiens COLLAGEN TYPE II - ALPHA 1 - NEAR N 12 Gly Val Lys1 5 amino acids amino acid linear peptide internal Homo sapiens COLLAGENTYPE II - ALPHA 1 -NEAR N2 13 Pro Gly Val Lys Gly 1 5 6 amino acidsamino acid linear peptide N-terminal Homo sapiens COLLAGEN TYPE III-ALPHA 1 - N TERM 14 Asp Val Lys Ser Gly Val 1 5 7 amino acids aminoacid linear peptide C-terminal Homo sapiens COLLAGEN TYPE III - ALPHA1 - C TERM 15 Glu Lys Ala Gly Gly Phe Ala 1 5 9 amino acids amino acidlinear peptide internal Homo sapiens COLLAGEN TYPE III - ALPHA 1 - NEARN 16 Gly Phe Pro Gly Met Lys Gly His Arg 1 5 9 amino acids amino acidlinear peptide internal Homo sapiens COLLAGEN TYPE III - ALPHA 1 - NEARC 17 Gly Ala Ala Gly Ile Lys Gly His Arg 1 5 1341 amino acids amino acidlinear protein Homo sapiens COLLAGEN ALPHA 1 (I) 18 Met Phe Ser Phe ValAsp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr 1 5 10 15 Ala Leu Leu ThrHis Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp 20 25 30 Glu Asp Ile ProPro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His 35 40 45 Asp Arg Asp ValTrp Lys Pro Glu Pro Cys Gln Ile Cys Val Cys Asp 50 55 60 Asn Gly Lys ValLeu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn 65 70 75 80 Cys Pro GlyAla Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro 85 90 95 Asp Gly SerGlu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly 100 105 110 Pro LysGly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro 115 120 125 ProGly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro 130 135 140Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala 145 150155 160 Pro Gln Leu Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser Val165 170 175 Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly ProPro 180 185 190 Gly Ala Pro Gly Pro Glx Gly Phe Glx Gly Pro Pro Gly GlxPro Gly 195 200 205 Glx Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly ProPro Gly Pro 210 215 220 Pro Gly Lys Asx Gly Asx Asx Gly Glx Ala Gly LysPro Gly Arg Pro 225 230 235 240 Gly Glx Arg Gly Pro Pro Gly Pro Glx GlyAla Arg Gly Leu Pro Gly 245 250 255 Thr Ala Gly Leu Pro Gly Met Lys GlyHis Arg Gly Phe Ser Gly Leu 260 265 270 Asx Gly Ala Lys Gly Asx Ala GlyPro Ala Gly Pro Lys Gly Glx Pro 275 280 285 Gly Ser Pro Gly Glx Asx GlyAla Pro Gly Glx Met Gly Pro Pro Gly 290 295 300 Pro Lys Gly Asn Ser GlyGlu Pro Gly Ala Pro Gly Ser Lys Gly Asp 305 310 315 320 Thr Gly Ala LysGly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro 325 330 335 Gly Pro AlaGly Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly 340 345 350 Pro ThrGly Leu Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser 355 360 365 ArgGly Phe Pro Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala 370 375 380Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly 385 390395 400 Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu405 410 415 Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly ProPro 420 425 430 Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly ProPro Gly 435 440 445 Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro Gly ProLys Gly Ala 450 455 460 Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly ValPro Gly Pro Pro 465 470 475 480 Gly Ala Val Gly Pro Ala Gly Lys Asp GlyGlu Ala Gly Ala Gln Gly 485 490 495 Pro Pro Gly Pro Ala Gly Pro Ala GlyGlu Arg Gly Glu Gln Gly Pro 500 505 510 Ala Gly Ser Pro Gly Phe Gln GlyLeu Pro Gly Pro Ala Gly Pro Pro 515 520 525 Gly Glu Ala Gly Lys Pro GlyGlu Gln Gly Val Pro Gly Asp Leu Gly 530 535 540 Ala Pro Gly Pro Ser GlyAla Arg Gly Glu Arg Gly Phe Pro Gly Glu 545 550 555 560 Arg Gly Val GlnGly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn 565 570 575 Gly Ala ProGly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly 580 585 590 Ala ProGly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu 595 600 605 ArgGly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala 610 615 620Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly 625 630635 640 Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp645 650 655 Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly AlaArg 660 665 670 Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly ProAla Gly 675 680 685 Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly AlaLys Gly Glu 690 695 700 Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly ProPro Gly Pro Ala 705 710 715 720 Gly Pro Ala Gly Pro Pro Gly Pro Ile GlyAsn Val Gly Ala Pro Gly 725 730 735 Ala Lys Gly Ala Arg Gly Ser Ala GlyPro Pro Gly Ala Thr Gly Phe 740 745 750 Pro Gly Ala Ala Gly Arg Val GlyPro Pro Gly Pro Ser Gly Asn Ala 755 760 765 Gly Pro Pro Gly Pro Pro GlyPro Ala Gly Lys Glu Gly Gly Lys Gly 770 775 780 Pro Arg Gly Glu Thr GlyPro Ala Gly Arg Pro Gly Glu Val Gly Pro 785 790 795 800 Pro Gly Pro ProGly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp 805 810 815 Gly Pro AlaGly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly 820 825 830 Gln ArgGly Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe 835 840 845 ProGly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser 850 855 860Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly 865 870875 880 Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala885 890 895 Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly AspArg 900 905 910 Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Xaa Gly AlaXaa Gly 915 920 925 Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly AspArg Gly Glu 930 935 940 Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly ProAla Gly Ala Arg 945 950 955 960 Gly Pro Ala Gly Pro Gln Gly Pro Arg GlyAsp Lys Gly Glu Thr Gly 965 970 975 Glu Gln Gly Asp Arg Gly Ile Lys GlyHis Arg Gly Phe Ser Gly Leu 980 985 990 Gln Gly Pro Pro Gly Pro Pro GlySer Pro Gly Glu Gln Gly Pro Ser 995 1000 1005 Gly Ala Ser Gly Pro AlaGly Pro Arg Gly Pro Pro Gly Ser Ala Gly 1010 1015 1020 Ala Pro Gly LysAsp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro 1025 1030 1035 1040 ProGly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro 1045 10501055 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe1060 1065 1070 Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala HisLys Gly 1075 1080 1085 Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Val ValArg Asp Arg Asp 1090 1095 1100 Leu Glu Val Asp Thr Thr Leu Lys Ser LeuSer Gln Gln Ile Glu Asn 1105 1110 1115 1120 Ile Arg Ser Pro Glu Gly XaaArg Lys Asn Pro Ala Arg Thr Cys Arg 1125 1130 1135 Asp Leu Lys Met CysHis Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile 1140 1145 1150 Asp Pro AsnGln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn 1155 1160 1165 MetGlu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala 1170 11751180 Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Asp Arg His1185 1190 1195 1200 Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln PheGlu Tyr Gly 1205 1210 1215 Gly Gln Gly Ser Asp Pro Ala Asp Val Ala IleGln Leu Thr Phe Leu 1220 1225 1230 Arg Leu Met Ser Thr Glu Ala Ser GlnAsn Ile Thr Tyr His Cys Lys 1235 1240 1245 Asn Ser Val Ala Tyr Met AspGln Gln Thr Gly Asn Leu Lys Lys Ala 1250 1255 1260 Leu Leu Leu Xaa GlySer Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn 1265 1270 1275 1280 Ser ArgPhe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His Thr 1285 1290 1295Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser 13001305 1310 Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala ProAsp 1315 1320 1325 Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu1330 1335 1340 1366 amino acids amino acid linear protein Homo sapienscollagen alpha 2- type I 19 Met Leu Ser Phe Val Asp Thr Arg Thr Leu LeuLeu Leu Ala Val Thr 1 5 10 15 Leu Cys Leu Ala Thr Cys Gln Ser Leu GlnGlu Glu Thr Val Arg Lys 20 25 30 Gly Pro Ala Gly Asp Arg Gly Pro Arg GlyGlu Arg Gly Pro Pro Gly 35 40 45 Pro Pro Gly Arg Asp Gly Glu Asp Gly ProThr Gly Pro Pro Gly Pro 50 55 60 Pro Gly Pro Pro Gly Pro Pro Gly Leu GlyGly Asn Phe Ala Ala Gln 65 70 75 80 Tyr Asp Gly Lys Gly Val Gly Leu GlyPro Gly Pro Met Gly Leu Met 85 90 95 Gly Pro Arg Gly Pro Pro Gly Ala AlaGly Ala Pro Gly Pro Gln Gly 100 105 110 Phe Gln Gly Pro Ala Gly Glu ProGly Glu Pro Gly Gln Thr Gly Pro 115 120 125 Ala Gly Ala Arg Gly Pro AlaGly Pro Pro Gly Lys Ala Gly Glu Asp 130 135 140 Gly His Pro Gly Lys ProGly Arg Pro Gly Glu Arg Gly Val Val Gly 145 150 155 160 Pro Gln Gly AlaArg Gly Phe Pro Gly Thr Pro Gly Leu Pro Gly Phe 165 170 175 Lys Gly IleArg Gly His Asn Gly Leu Asp Gly Leu Lys Gly Gln Pro 180 185 190 Gly AlaPro Gly Val Lys Gly Glu Pro Gly Ala Pro Gly Glu Asn Gly 195 200 205 ThrPro Gly Gln Thr Gly Ala Arg Gly Leu Pro Gly Glu Arg Gly Arg 210 215 220Val Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Ser Asp Gly Ser Val 225 230235 240 Gly Pro Val Gly Pro Ala Gly Pro Asn Gly Ser Ala Gly Pro Pro Gly245 250 255 Phe Pro Gly Ala Pro Gly Pro Lys Gly Glu Ile Gly Ala Val GlyAsn 260 265 270 Ala Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly Glu Val GlyLeu Pro 275 280 285 Gly Leu Ser Gly Pro Val Gly Pro Pro Gly Asn Pro GlyAla Asn Gly 290 295 300 Leu Thr Gly Ala Lys Gly Ala Ala Gly Leu Pro GlyVal Ala Gly Ala 305 310 315 320 Pro Gly Leu Pro Gly Pro Arg Gly Ile ProGly Pro Pro Gly Ala Ala 325 330 335 Gly Thr Thr Gly Ala Arg Gly Leu ValGly Glu Pro Gly Pro Ala Gly 340 345 350 Ser Lys Gly Glu Ser Gly Asn LysGly Glu Pro Gly Ser Ala Gly Pro 355 360 365 Gln Gly Pro Pro Gly Pro SerGly Glu Glu Gly Lys Arg Gly Pro Asn 370 375 380 Gly Glu Ala Gly Ser AlaGly Pro Pro Gly Pro Pro Gly Leu Arg Gly 385 390 395 400 Ser Pro Gly SerArg Gly Leu Pro Gly Ala Asp Gly Arg Ala Gly Val 405 410 415 Met Gly ProPro Gly Ser Arg Gly Ala Ser Gly Pro Ala Gly Val Arg 420 425 430 Gly ProAsn Gly Asp Ala Gly Arg Pro Gly Glu Pro Gly Leu Met Gly 435 440 445 ProArg Gly Leu Pro Gly Ser Pro Gly Asn Ile Gly Pro Ala Gly Lys 450 455 460Glu Gly Pro Val Gly Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile 465 470475 480 Gly Pro Val Gly Ala Arg Gly Glu Pro Gly Asn Ile Gly Phe Pro Gly485 490 495 Pro Lys Gly Pro Thr Gly Asp Pro Gly Lys Asn Gly Asp Lys GlyHis 500 505 510 Ala Gly Leu Ala Gly Ala Arg Gly Ala Pro Gly Pro Asp GlyAsn Asn 515 520 525 Gly Ala Gln Gly Pro Pro Gly Pro Gln Gly Val Gln GlyGly Lys Gly 530 535 540 Glu Gln Gly Pro Ala Gly Pro Pro Gly Phe Gln GlyLeu Pro Gly Pro 545 550 555 560 Ser Gly Pro Ala Gly Glu Val Gly Lys ProGly Glu Arg Gly Leu His 565 570 575 Gly Glu Phe Gly Leu Pro Gly Pro AlaGly Pro Arg Gly Glu Arg Gly 580 585 590 Pro Pro Gly Glu Ser Gly Ala AlaGly Pro Thr Gly Pro Ile Gly Ser 595 600 605 Arg Gly Pro Ser Gly Pro ProGly Pro Asp Gly Asn Lys Gly Glu Pro 610 615 620 Gly Val Val Gly Ala ValGly Thr Ala Gly Pro Ser Gly Pro Ser Gly 625 630 635 640 Leu Pro Gly GluArg Gly Ala Ala Gly Ile Pro Gly Gly Lys Gly Glu 645 650 655 Lys Gly GluPro Gly Leu Arg Gly Glu Ile Gly Asn Pro Gly Arg Asp 660 665 670 Gly AlaArg Gly Ala His Gly Ala Val Gly Ala Pro Gly Pro Ala Gly 675 680 685 AlaThr Gly Asp Arg Gly Glu Ala Gly Ala Ala Gly Pro Ala Gly Pro 690 695 700Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Val Gly Pro Ala 705 710715 720 Gly Pro Asn Gly Phe Ala Gly Pro Ala Gly Ala Ala Gly Gln Pro Gly725 730 735 Ala Lys Gly Glu Arg Gly Gly Lys Gly Pro Lys Gly Glu Asn GlyVal 740 745 750 Val Gly Pro Thr Gly Pro Val Gly Ala Ala Gly Pro Ala GlyPro Asn 755 760 765 Gly Pro Pro Gly Pro Ala Gly Ser Arg Gly Asp Gly GlyPro Pro Gly 770 775 780 Met Thr Gly Phe Pro Gly Ala Ala Gly Arg Thr GlyPro Pro Gly Pro 785 790 795 800 Ser Gly Ile Ser Gly Pro Pro Gly Pro ProGly Pro Ala Gly Lys Glu 805 810 815 Gly Leu Arg Gly Pro Arg Gly Asp GlnGly Pro Val Gly Arg Thr Gly 820 825 830 Glu Val Gly Ala Val Gly Pro ProGly Phe Ala Gly Glu Lys Gly Pro 835 840 845 Ser Gly Glu Ala Gly Thr AlaGly Pro Pro Gly Thr Pro Gly Pro Gln 850 855 860 Gly Leu Leu Gly Ala ProGly Ile Leu Gly Leu Pro Gly Ser Arg Gly 865 870 875 880 Glu Arg Gly LeuPro Gly Val Ala Gly Ala Val Gly Glu Pro Gly Pro 885 890 895 Leu Gly IleAla Gly Pro Pro Gly Ala Arg Gly Pro Pro Gly Ala Val 900 905 910 Gly SerPro Gly Val Asn Gly Ala Pro Gly Glu Ala Gly Arg Asp Gly 915 920 925 AsnPro Gly Asn Asp Gly Pro Pro Gly Arg Asp Gly Gln Pro Gly His 930 935 940Lys Gly Glu Arg Gly Tyr Pro Gly Asn Ile Gly Pro Val Gly Ala Ala 945 950955 960 Gly Ala Pro Gly Pro His Gly Pro Val Gly Pro Ala Gly Lys His Gly965 970 975 Asn Arg Gly Glu Thr Gly Pro Ser Gly Pro Val Gly Pro Ala GlyAla 980 985 990 Val Gly Pro Arg Gly Pro Ser Gly Pro Gln Gly Ile Arg GlyAsp Lys 995 1000 1005 Gly Glu Pro Gly Glu Lys Gly Pro Arg Gly Leu ProGly Phe Lys Gly 1010 1015 1020 His Asn Gly Leu Gln Gly Leu Pro Gly IleAla Gly His His Gly Asp 1025 1030 1035 1040 Gln Gly Ala Pro Gly Ser ValGly Pro Ala Gly Pro Arg Gly Pro Ala 1045 1050 1055 Gly Pro Ser Gly ProAla Gly Lys Asp Gly Arg Thr Gly His Pro Gly 1060 1065 1070 Thr Val GlyPro Ala Gly Ile Arg Gly Pro Gln Gly His Gln Gly Pro 1075 1080 1085 AlaGly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Val Ser 1090 10951100 Gly Gly Gly Tyr Asp Phe Gly Tyr Asp Gly Asp Phe Tyr Arg Ala Asp1105 1110 1115 1120 Gln Pro Arg Ser Ala Pro Ser Leu Arg Pro Lys Asp TyrGlu Val Asp 1125 1130 1135 Ala Thr Leu Lys Ser Leu Asn Asn Gln Ile GluThr Leu Leu Thr Pro 1140 1145 1150 Glu Gly Ser Arg Lys Asn Pro Ala ArgThr Cys Arg Asp Leu Arg Leu 1155 1160 1165 Ser His Pro Glu Trp Ser SerGly Tyr Tyr Trp Ile Asp Pro Asn Gln 1170 1175 1180 Gly Cys Thr Met GluAla Ile Lys Val Tyr Cys Asp Phe Pro Thr Gly 1185 1190 1195 1200 Glu ThrCys Ile Arg Ala Gln Pro Glu Asn Ile Pro Ala Lys Asn Trp 1205 1210 1215Tyr Arg Ser Ser Lys Asp Lys Lys His Val Trp Leu Gly Glu Thr Ile 12201225 1230 Asn Ala Gly Ser Gln Phe Glu Tyr Asn Val Glu Gly Val Thr SerLys 1235 1240 1245 Glu Met Ala Thr Gln Leu Ala Phe Met Arg Leu Leu AlaAsn Tyr Ala 1250 1255 1260 Ser Gln Asn Ile Thr Tyr His Cys Lys Asn SerIle Ala Tyr Met Asp 1265 1270 1275 1280 Glu Glu Thr Gly Asn Leu Lys LysAla Val Ile Leu Gln Gly Ser Asn 1285 1290 1295 Asp Val Glu Leu Val AlaGlu Gly Asn Ser Arg Phe Thr Tyr Thr Val 1300 1305 1310 Leu Val Asp GlyCys Ser Lys Lys Thr Asn Glu Trp Gly Lys Thr Ile 1315 1320 1325 Ile GluTyr Lys Thr Asn Lys Pro Ser Arg Leu Pro Phe Leu Asp Ile 1330 1335 1340Ala Pro Leu Asp Ile Gly Gly Ala Asp His Glu Phe Phe Val Asp Ile 13451350 1355 1360 Gly Pro Val Cys Phe Lys 1365 1418 amino acids amino acidlinear protein Homo sapiens COLLAGEN -ALPHA 1 (II) 20 Met Ile Arg LeuGly Ala Pro Gln Ser Leu Val Leu Leu Thr Leu Leu 1 5 10 15 Val Ala AlaVal Leu Arg Cys Gln Gly Gln Asp Val Arg Gln Pro Gly 20 25 30 Pro Lys GlyGln Lys Gly Glu Pro Gly Asp Ile Lys Asp Ile Val Gly 35 40 45 Pro Lys GlyPro Pro Gly Pro Gln Gly Pro Ala Gly Glu Gln Gly Pro 50 55 60 Arg Gly AspArg Gly Asp Lys Gly Glu Lys Gly Ala Pro Gly Pro Arg 65 70 75 80 Gly ArgAsp Gly Glu Pro Gly Thr Leu Gly Asn Pro Gly Pro Pro Gly 85 90 95 Pro ProGly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Ala 100 105 110 GlnMet Ala Gly Gly Phe Asp Glu Lys Ala Gly Gly Ala Gln Leu Gly 115 120 125Val Met Gln Gly Pro Met Gly Pro Met Gly Pro Arg Gly Pro Pro Gly 130 135140 Pro Ala Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Asn Pro Gly Glu 145150 155 160 Pro Gly Glu Pro Gly Val Ser Gly Pro Met Gly Pro Arg Gly ProPro 165 170 175 Gly Pro Pro Gly Lys Pro Gly Asp Asp Gly Glu Ala Gly LysPro Gly 180 185 190 Lys Ala Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly AlaArg Gly Phe 195 200 205 Pro Gly Thr Pro Gly Leu Pro Gly Val Lys Gly HisArg Gly Tyr Pro 210 215 220 Gly Leu Asp Gly Ala Lys Gly Glu Ala Gly AlaPro Gly Val Lys Gly 225 230 235 240 Glu Ser Gly Ser Pro Gly Glu Asn GlySer Pro Gly Pro Met Gly Pro 245 250 255 Arg Gly Leu Pro Gly Glu Arg GlyArg Thr Gly Pro Ala Gly Ala Ala 260 265 270 Gly Ala Arg Gly Asn Asp GlyGln Pro Gly Pro Ala Gly Pro Pro Gly 275 280 285 Pro Val Gly Pro Ala GlyGly Pro Gly Phe Pro Gly Ala Pro Gly Ala 290 295 300 Lys Gly Glu Ala GlyPro Thr Gly Ala Arg Gly Pro Glu Gly Ala Gln 305 310 315 320 Gly Pro ArgGly Glu Pro Gly Thr Pro Gly Ser Pro Gly Pro Ala Gly 325 330 335 Ala SerGly Asn Pro Gly Thr Asp Gly Ile Pro Gly Ala Lys Gly Ser 340 345 350 AlaGly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Pro Arg 355 360 365Gly Pro Pro Asp Pro Gln Gly Ala Thr Gly Pro Leu Gly Pro Lys Gly 370 375380 Gln Thr Gly Lys Pro Gly Ile Ala Gly Phe Lys Gly Glu Gln Gly Pro 385390 395 400 Lys Gly Glu Pro Gly Pro Ala Gly Pro Gln Gly Ala Pro Gly ProAla 405 410 415 Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly GlyVal Gly 420 425 430 Pro Ile Gly Pro Pro Gly Glu Arg Gly Ala Pro Gly AsnArg Gly Phe 435 440 445 Pro Gly Gln Asp Gly Leu Ala Gly Pro Lys Gly AlaPro Gly Glu Arg 450 455 460 Gly Pro Ser Gly Leu Ala Gly Pro Lys Gly AlaAsn Gly Asp Pro Gly 465 470 475 480 Arg Pro Gly Glu Pro Gly Leu Pro GlyAla Arg Gly Leu Thr Gly Arg 485 490 495 Pro Gly Asp Ala Gly Pro Gln GlyLys Val Gly Pro Ser Gly Ala Pro 500 505 510 Gly Glu Asp Gly Arg Pro GlyPro Pro Gly Pro Gln Gly Ala Arg Gly 515 520 525 Gln Pro Gly Val Met GlyPhe Pro Gly Pro Lys Gly Ala Asn Gly Glu 530 535 540 Pro Gly Lys Ala GlyGlu Lys Gly Leu Pro Gly Ala Pro Gly Leu Arg 545 550 555 560 Gly Leu ProGly Lys Asp Gly Glu Thr Gly Ala Glu Gly Pro Pro Gly 565 570 575 Pro AlaGly Pro Ala Gly Glu Arg Gly Glu Gln Gly Ala Pro Gly Pro 580 585 590 SerGly Phe Gln Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Glu Gly 595 600 605Gly Lys Pro Gly Asp Gln Gly Val Pro Gly Glu Ala Gly Ala Pro Gly 610 615620 Leu Val Gly Pro Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Ser 625630 635 640 Pro Gly Ala Gln Gly Leu Gln Gly Pro Arg Gly Leu Pro Gly ThrPro 645 650 655 Gly Thr Asp Gly Pro Lys Gly Ala Ser Gly Pro Ala Gly ProPro Gly 660 665 670 Ala Gln Gly Pro Pro Gly Leu Gln Gly Met Pro Gly GluArg Gly Ala 675 680 685 Ala Gly Ile Ala Gly Pro Lys Gly Asp Arg Gly AspVal Gly Glu Lys 690 695 700 Gly Pro Glu Gly Ala Pro Gly Lys Asp Gly GlyArg Gly Leu Thr Gly 705 710 715 720 Pro Ile Gly Pro Pro Gly Pro Ala GlyAla Asn Gly Glu Lys Gly Glu 725 730 735 Val Gly Pro Pro Gly Pro Ala GlySer Ala Gly Ala Arg Gly Ala Pro 740 745 750 Gly Glu Arg Gly Glu Thr GlyPro Pro Gly Thr Ser Gly Ile Ala Gly 755 760 765 Pro Pro Gly Ala Asp GlyGln Pro Gly Ala Lys Gly Glu Gln Gly Glu 770 775 780 Ala Gly Gln Lys GlyAsp Ala Gly Ala Pro Gly Pro Gln Gly Pro Ser 785 790 795 800 Gly Ala ProGly Pro Gln Gly Pro Thr Gly Val Thr Gly Pro Lys Gly 805 810 815 Ala ArgGly Ala Gln Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala 820 825 830 AlaGly Arg Val Gly Pro Pro Gly Ser Asn Gly Asn Pro Gly Pro Pro 835 840 845Gly Pro Pro Gly Pro Ser Gly Lys Asp Gly Pro Lys Gly Ala Arg Gly 850 855860 Asp Ser Gly Pro Pro Gly Arg Ala Gly Glu Pro Gly Leu Gln Gly Pro 865870 875 880 Ala Gly Pro Pro Gly Glu Lys Gly Glu Pro Gly Asp Asp Gly ProSer 885 890 895 Gly Ala Glu Gly Pro Pro Gly Pro Gln Gly Leu Ala Gly GlnArg Gly 900 905 910 Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly PhePro Gly Leu 915 920 925 Pro Gly Pro Ser Gly Glu Pro Gly Gln Gln Gly AlaPro Gly Ala Ser 930 935 940 Gly Asp Arg Gly Pro Pro Gly Pro Val Gly ProPro Gly Leu Thr Gly 945 950 955 960 Pro Ala Gly Glu Pro Gly Arg Glu GlySer Pro Gly Ala Asp Gly Pro 965 970 975 Pro Gly Arg Asp Gly Ala Ala GlyVal Lys Gly Asp Arg Gly Glu Thr 980 985 990 Gly Ala Val Gly Ala Pro GlyAla Pro Gly Pro Pro Gly Ser Pro Gly 995 1000 1005 Pro Ala Gly Pro ThrGly Lys Gln Gly Asp Arg Gly Glu Ala Gly Ala 1010 1015 1020 Gln Gly ProMet Gly Pro Ser Gly Pro Ala Gly Ala Arg Gly Ile Gln 1025 1030 1035 1040Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Ala Gly Glu Pro Gly 10451050 1055 Glu Arg Gly Leu Lys Gly His Arg Gly Phe Thr Gly Leu Gln GlyLeu 1060 1065 1070 Pro Gly Pro Pro Gly Pro Ser Gly Asp Gln Gly Ala SerGly Pro Ala 1075 1080 1085 Gly Pro Ser Gly Pro Arg Gly Pro Pro Gly ProVal Gly Pro Ser Gly 1090 1095 1100 Lys Asp Gly Ala Asn Gly Ile Pro GlyPro Ile Gly Pro Pro Gly Pro 1105 1110 1115 1120 Arg Gly Arg Ser Gly GluThr Gly Pro Ala Gly Pro Pro Gly Asn Pro 1125 1130 1135 Gly Pro Pro GlyPro Pro Gly Pro Pro Gly Pro Gly Ile Asp Met Ser 1140 1145 1150 Ala PheAla Gly Leu Gly Pro Arg Glu Lys Gly Pro Asp Pro Leu Gln 1155 1160 1165Tyr Met Arg Ala Asp Gln Ala Ala Gly Gly Leu Arg Gln His Asp Ala 11701175 1180 Glu Val Asp Ala Thr Leu Lys Ser Leu Asn Asn Gln Ile Glu SerIle 1185 1190 1195 1200 Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala ArgThr Cys Arg Asp 1205 1210 1215 Leu Lys Leu Cys His Pro Glu Trp Lys SerGly Asp Tyr Trp Ile Asp 1220 1225 1230 Pro Asn Gln Gly Cys Thr Leu AspAla Met Lys Val Phe Cys Asn Met 1235 1240 1245 Glu Thr Gly Glu Thr CysVal Tyr Pro Asn Pro Ala Asn Val Pro Lys 1250 1255 1260 Lys Asn Trp TrpSer Ser Lys Ser Lys Glu Lys Lys His Ile Trp Phe 1265 1270 1275 1280 GlyGlu Thr Ile Asn Gly Gly Phe His Phe Ser Tyr Gly Asp Asp Asn 1285 12901295 Leu Ala Pro Asn Thr Ala Asn Val Gln Met Thr Phe Leu Arg Leu Leu1300 1305 1310 Ser Thr Glu Gly Ser Gln Asn Ile Thr Tyr His Cys Lys AsnSer Ile 1315 1320 1325 Ala Tyr Leu Asp Glu Ala Ala Gly Asn Leu Lys LysAla Leu Leu Ile 1330 1335 1340 Gln Gly Ser Asn Asp Val Glu Ile Arg AlaGlu Gly Asn Ser Arg Phe 1345 1350 1355 1360 Thr Tyr Thr Ala Leu Lys AspGly Cys Thr Lys His Thr Gly Lys Trp 1365 1370 1375 Gly Lys Thr Val IleGlu Tyr Arg Ser Gln Lys Thr Ser Arg Leu Pro 1380 1385 1390 Ile Ile AspIle Ala Pro Met Asp Ile Gly Gly Pro Glu Gln Glu Phe 1395 1400 1405 GlyVal Asp Ile Gly Pro Val Cys Phe Leu 1410 1415 1078 amino acids aminoacid linear protein Homo sapiens COLLAGEN ALPHA 1 (III) 21 Gln Asn TyrSer Pro Gln Tyr Asp Ser Tyr Asp Val Lys Ser Gly Gly 1 5 10 15 Val AlaVal Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala Gly Pro Pro 20 25 30 Gly ProPro Gly Pro Pro Gly Thr Ser Gly His Pro Gly Ser Pro Gly 35 40 45 Ser ProGly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro 50 55 60 Ser GlyPro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala 65 70 75 80 GlyLys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Asp Arg Gly 85 90 95 LeuPro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe 100 105 110Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys 115 120125 Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly 130135 140 Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu145 150 155 160 Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg GlyAsn Asp 165 170 175 Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro GlyPro Pro Gly 180 185 190 Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys GlyGlu Val Gly Pro 195 200 205 Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro GlyGln Arg Gly Glu Pro 210 215 220 Gly Pro Gln Gly His Ala Gly Ala Gln GlyPro Pro Gly Pro Pro Gly 225 230 235 240 Ile Asn Gly Ser Pro Gly Gly LysGly Glu Met Gly Pro Ala Gly Ile 245 250 255 Pro Gly Ala Pro Gly Leu MetGly Ala Arg Gly Pro Pro Gly Pro Ala 260 265 270 Gly Ala Asn Gly Ala ProGly Leu Arg Gly Gly Ala Gly Glu Pro Gly 275 280 285 Lys Asn Gly Ala LysGly Glu Pro Gly Pro Arg Gly Glu Arg Gly Glu 290 295 300 Ala Gly Ile ProGly Val Pro Gly Ala Lys Gly Glu Asp Gly Lys Asp 305 310 315 320 Gly SerPro Gly Asp Pro Gly Ala Asn Gly Leu Pro Gly Ala Ala Gly 325 330 335 GluArg Gly Ala Leu Gly Ser Arg Gly Pro Ala Gly Pro Asn Gly Ile 340 345 350Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Pro Ala 355 360365 Gly Pro Arg Gly Ala Ala Gly Glu Pro Gly Arg Asp Gly Val Pro Gly 370375 380 Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly Pro Gly Ser385 390 395 400 Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Ser GlyArg Pro 405 410 415 Gly Pro Pro Gly Pro Ser Gly Pro Arg Gly Gln Pro GlyVal Met Gly 420 425 430 Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro GlyLys Asn Gly Glu 435 440 445 Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln GlyPro Pro Gly Lys Asn 450 455 460 Gly Glu Tyr Gly Pro Gln Gly Pro Pro GlyPro Thr Gly Pro Gly Gly 465 470 475 480 Asp Lys Gly Asp Thr Gly Pro ArgGly Pro Gln Gly Leu Gln Gly Leu 485 490 495 Pro Gly Thr Gly Gly Pro ProGly Glu Asn Glu Lys Pro Gly Glu Pro 500 505 510 Gly Pro Lys Gly Glu AlaGly Ala Pro Gly Ala Pro Gly Gly Lys Gly 515 520 525 Asp Ala Gly Ala ProGly Glu Arg Gly Pro Pro Gly Leu Ala Gly Ala 530 535 540 Pro Gly Leu ArgGly Gly Ala Gly Pro Pro Gly Pro Glu Gly Gly Lys 545 550 555 560 Gly AlaAla Gly Pro Pro Gly Pro Pro Gly Ala Ala Gly Thr Pro Gly 565 570 575 LeuGln Gly Met Pro Gly Glu Arg Gly Gly Leu Gly Ser Pro Gly Pro 580 585 590Lys Gly Asp Lys Gly Glu Pro Gly Gly Pro Gly Ala Asp Gly Val Pro 595 600605 Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly Pro Pro Gly 610615 620 Pro Ala Gly Gln Pro Gly Asp Lys Gly Glu Gly Gly Ala Pro Gly Leu625 630 635 640 Pro Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg GlyGlu Thr 645 650 655 Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro GlyGln Asn Gly 660 665 670 Glu Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro GlyGlu Lys Gly Glu 675 680 685 Gly Gly Pro Pro Gly Val Ala Val Pro Pro GlyGly Ser Gly Pro Ala 690 695 700 Gly Pro Pro Gly Pro Gln Gly Val Lys GlyGlu Arg Gly Ser Pro Gly 705 710 715 720 Gly Pro Gly Ala Ala Gly Phe ProGly Ala Arg Gly Leu Pro Gly Pro 725 730 735 Pro Gly Ser Asn Gly Asn ProGly Pro Pro Gly Pro Ser Gly Ser Pro 740 745 750 Gly Lys Asp Gly Pro ProGly Pro Ala Gly Asn Thr Gly Ala Pro Gly 755 760 765 Ser Pro Gly Val SerGly Pro Lys Gly Asp Ala Gly Gln Pro Gly Glu 770 775 780 Lys Gly Ser ProGly Ala Gln Gly Pro Pro Gly Ala Pro Gly Pro Leu 785 790 795 800 Gly IleAla Gly Ile Thr Gly Ala Arg Gly Leu Ala Gly Pro Pro Gly 805 810 815 MetPro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gly Val Lys Gly Glu 820 825 830Ser Gly Lys Pro Gly Ala Asn Gly Leu Ser Gly Glu Arg Gly Pro Pro 835 840845 Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Ala Gly Glu Pro Gly 850855 860 Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly Arg Asp Gly Ser865 870 875 880 Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Ser Pro GlyAla Pro 885 890 895 Gly Ala Pro Gly His Pro Gly Pro Pro Gly Pro Val GlyPro Ala Gly 900 905 910 Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala GlyPro Ala Gly Ala 915 920 925 Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro GlyPro Gln Gly Pro Arg 930 935 940 Gly Asp Lys Gly Glu Thr Gly Glu Arg GlyAla Ala Gly Ile Lys Gly 945 950 955 960 His Arg Gly Phe Pro Gly Asn ProGly Ala Pro Gly Ser Pro Gly Pro 965 970 975 Ala Gly Gln Gln Gly Ala IleGly Ser Pro Gly Pro Ala Gly Pro Arg 980 985 990 Gly Pro Val Gly Pro SerGly Pro Pro Gly Lys Asp Gly Thr Ser Gly 995 1000 1005 His Pro Gly ProIle Gly Pro Pro Gly Pro Arg Gly Asn Arg Gly Glu 1010 1015 1020 Arg GlySer Glu Gly Ser Pro Gly His Pro Gly Gln Ser Gly Pro Pro 1025 1030 10351040 Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val Gly Ala Pro1045 1050 1055 Ala Ile Ala Gly Ile Gly Ala Glu Lys Ala Gly Gly Phe AlaPro Tyr 1060 1065 1070 Tyr Gly Asp Glu Pro Met 1075

What is claimed is:
 1. A method of determining collagen fragments in abody fluid, wherein a sample of said body fluid is brought into contactwith at least one immunological binding partner for the collagenfragments, said binding partner being an antibody immunoreactive withsynthetic peptides the sequences of which are essentially derived fromcollagen and containing potential sites for cross-linking, saidimmunological binding partners being incorporated, either as wholeantibodies or as immunologically active fragments thereof, in an assayfor quantitative determination of collagen fragments in the sample.
 2. Amethod according to claim 1, wherein the sample of body fluid inaddition to the contacting with the immunological binding partner(s) isbrought into direct contact with the corresponding synthetic peptidecontaining potential sites for cross-linking.
 3. A method according toclaim 1, wherein the immunological binding partner has been raised byimmunization with the corresponding synthetic peptide containingpotential sites for cross-linking.
 4. A method according to claim 2,wherein the immunological binding partner has been raised byimmunization with the corresponding synthetic peptide containingpotential sites for cross-linking.
 5. A method according to claim 1,wherein the potential sites for cross-linking comprise lysine orhydroxylysine residues incorporated in the peptide sequence.
 6. A methodaccording to claim 1, wherein the sequence of the synthetic peptide isderived from the sequence of human collagen type I and consistingessentially of one of the following structuresAsp-Glu-Lys-Ser-Thr-Gly-Gly Glu-Lys-Ala-His-Asp-Gly-Gly-ArgGln-Tyr-Asp-Gly-Lys-Gly-Val-Gly Gly-Met-Lys-Gly-His-ArgGly-Ile-Lys-Gly-His-Arg Gly-Phe-Lys-Gly-Ile-Arg orGly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn.
 7. A method according to claim 2,wherein the sequence of the synthetic peptide is derived from thesequence of human collagen type I and consisting essentially of one ofthe following structures Asp-Glu-Lys-Ser-Thr-Gly-GlyGlu-Lys-Ala-His-Asp-Gly-Gly-Arg Gln-Tyr-Asp-Gly-Lys-Gly-Val-GlyGly-Met-Lys-Gly-His-Arg Gly-Ile-Lys-Gly-His-Arg Gly-Phe-Lys-Gly-Ile-Argor Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn.
 8. A method according to claim1, wherein the sequence of the synthetic peptide is derived from thesequence of human collagen type II and consisting essentially of one ofthe following structures Glu-Lys-Gly-Pro-Asp Gly-Val-LysPro-Gly-Val-Lys-Gly Pro-Gly-Pro-Lys-Gly-Glu Gly-Gln-Lys-Gly-Glu-Pro orGly-Asp-Ile-Lys-Asp-Ile-Val.
 9. A method according to claim 2, whereinthe sequence of the synthetic peptide is derived from the sequence ofhuman collagen type II and consisting essentially of one of thefollowing structures Glu-Lys-Gly-Pro-Asp Gly-Val-Lys Pro-Gly-Val-Lys-GlyPro-Gly-Pro-Lys-Gly-Glu Gly-Gln-Lys-Gly-Glu-Pro orGly-Asp-Ile-Lys-Asp-Ile-Val.
 10. A method according to claim 1, whereinthe sequence of the synthetic peptide is derived from the sequence ofhuman collagen type III and consisting essentially of one of thefollowing structures: Asp-Val-Lys-Ser-Gly-ValGlu-Lys-Ala-Gly-Gly-Phe-Ala Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg orGly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg.
 11. A method according to claim 2,wherein the sequence of the synthetic peptide is derived from thesequence of human collagen type III and consisting essentially of one ofthe following structures: Asp-Val-Lys-Ser-Gly-ValGlu-Lys-Ala-Gly-Gly-Phe-Ala Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg orGly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg.
 12. A method according to claim 1,wherein the body fluid is human urine, blood, serum or synovial fluid.13. A cell line that produces a monoclonal antibody binding to any ofthe synthetic peptides used in the method according to claim
 1. 14. Acell line that produces a monoclonal antibody binding to any of thesynthetic peptides used in the method according to claim
 2. 15. Amonoclonal antibody produced by the cell line according to claim
 13. 16.An immunological binding partner raised by immunization with syntheticpeptides selected from the group consisting of sequences derived fromthe sequence of human collagen type I and consisting essentially of oneof the following structures Asp-Glu-Lys-Ser-Thr-Gly-GlyGlu-Lys-Ala-His-Asp-Gly-Gly-Arg Gln-Tyr-Asp-Gly-Lys-Gly-Val-GlyGly-Met-Lys-Gly-His-Arg Gly-Ile-Lys-Gly-His-Arg Gly-Phe-Lys-Gly-Ile-Argand Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn, sequences derived from thesequence of human collagen type II and consisting essentially of one ofthe following structures Glu-Lys-Gly-Pro-Asp Gly-Val-LysPro-Gly-Val-Lys-Gly Pro-Gly-Pro-Lys-Gly-Glu Gly-Gln-Lys-Gly-Glu-Pro andGly-Asp-Ile-Lys-Asp-Ile-Val, and sequences derived from the sequence ofhuman collagen type III and consisting essentially of one of thefollowing structures: Asp-Val-Lys-Ser-Gly-ValGlu-Lys-Ala-Gly-Gly-Phe-Ala Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg andGly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg, and said polyclonal antibody beingused for the preparation of an immunological binding partner.
 17. A testkit for quantitating the amount of collagen fragments in a body fluid,said kit comprising at least one immunological binding partnerimmunoreactive with a synthetic peptide having a sequence essentiallyderived from collagen and containing one or more potential sites forcross-linking.
 18. A method of diagnosing the presence of disordersassociated with the metabolism of collagen in a patient, said methodcomprising the steps of: (a) contacting a sample of body fluid collectedfrom said patient with at least one immunological binding partner forcollagen fragments, said binding partner being an antibody or a fragmentthereof which is immunoreactive with synthetic peptides the sequences ofwhich are essentially derived from collagen and containing potentialsites for cross-linking, (b) detecting the quantity of said antibodybound to collagen fragments in the sample, and (c) comparing saidquantity to a previously established standard based on binding of saidantibody in samples originating from control subjects not suffering fromany disorder associated with the metabolism of collagen.
 19. A methodaccording to claim 18, wherein the antibody has been raised byimmunization with the corresponding synthetic peptide containingpotential sites for cross-linking.
 20. The method according to claim 18,which includes combining the determination of collagen fragments withother markers of the metabolism of bone tissue, connective tissue andother tissues in order to diagnose a given disorder.
 21. A method fordetermining whether a susceptible subject is suffering from collagendegradation comprising the steps of: (a) contacting a sample of bodyfluid collected from said subject with an antibody that is specificallyimmunochemically reactive with an antigenic determinant of saidcollagen, said antigenic determinant consisting essentially of (i) apotentially crosslinkable site of the molecule of said collagen or (ii)an antigenic determinant essentially immediately adjacent to apotentially crosslinkable site on said collagen molecule; (b) detectingthe quantity of said antibody bound to collagen fragments in saidsample; and (c) comparing said quantity to a previously establishedstandard based on binding of said antibody in samples from controlsubjects not suffering from collagen degradation.
 22. A method fordetermining the presence of collagen degradation products in a bodyfluid comprising the steps of: (a) contacting a sample of said bodyfluid with an antibody that is specifically immunochemically reactivewith an antigenic determinant of said collagen, said antigenicdeterminant consisting essentially of (i) a potentially crosslinkablesite of the molecule of said collagen or (ii) an antigenic determinantessentially immediately adjacent to a potentially crosslinkable site onsaid collagen molecule; and (b) detecting the quantity of said antibodybound to collagen fragments in said sample.